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Keywords:

  • lactic acid bacteria;
  • fermentation;
  • metabolism;
  • comparative genomic analysis;
  • virulence

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Central metabolism
  5. The effect of oxygen on metabolism of Streptococcaceae
  6. Proton motive force generation by the F1Fo ATPase and solute transport
  7. ADI pathway
  8. Evidence from genome sequences for adaptation of Streptococcaceae to nutrient-rich environments
  9. Future perspectives
  10. References

Lactic acid bacteria (LAB) are indigenous to food-related habitats as well as associated with the mucosal surfaces of animals. The LAB family Streptococcaceae consists of the genera Lactococcus and Streptococcus. Members of the family include the industrially important species Lactococcus lactis, which has a long history safe use in the fermentative food industry, and the disease-causing streptococci Streptococcus pneumoniae and Streptococcus pyogenes. The central metabolic pathways of the Streptococcaceae family have been extensively studied because of their relevance in the industrial use of some species, as well as their influence on virulence of others. Recent developments in high-throughput proteomic and DNA-microarray techniques, in in vivo NMR studies, and importantly in whole-genome sequencing have resulted in new insights into the metabolism of the Streptococcaceae family. The development of cost-effective high-throughput sequencing has resulted in the publication of numerous whole-genome sequences of lactococcal and streptococcal species. Comparative genomic analysis of these closely related but environmentally diverse species provides insight into the evolution of this family of LAB and shows that the relatively small genomes of members of the Streptococcaceae family have been largely shaped by the nutritionally rich environments they inhabit.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Central metabolism
  5. The effect of oxygen on metabolism of Streptococcaceae
  6. Proton motive force generation by the F1Fo ATPase and solute transport
  7. ADI pathway
  8. Evidence from genome sequences for adaptation of Streptococcaceae to nutrient-rich environments
  9. Future perspectives
  10. References

Lactic acid bacteria (LAB) are indigenous to food-related habitats as well as associated with the mucosal surfaces of animals (Teuber, 1995; Wood & Warner, 2003). LAB have long been used in the production of dairy, plant, and meat fermented food products, contributing to the taste and texture of the food products. They prevent spoilage by lowering the pH of the product and/or producing growth-inhibiting bacteriocins (Cotter et al., 2005). The use of LAB in milk fermentation is an ancient process dating back to the Early Neolithic period (Evershed et al., 2008). Many LAB species are also used as probiotics for human and animal consumption (Gilliland, 1989) and have generally regarded as safe (GRAS) Food and Drug Administration (FDA) status. In addition, most LAB are quite acid resistant and certain strains are able to effectively survive passage through the gastrointestinal (GI) tract, making LAB attractive potential delivery vehicles for recombinant protein vaccines (Medina et al., 2010). However, not all LAB species have a positive impact on food production or human health. Some can cause food spoilage, while several important (human) pathogenic species are included in the large clade of the LAB.

As their name suggests, lactic acid is the main end product of sugar fermentation in this group of microorganisms. While there are many bacteria that produce lactic acid, the umbrella-term LAB are generally restricted to the genera Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus, and Weissella (Holzapfel & Wood, 1995; Vandamme et al., 1996). The classification of LAB remains an unresolved issue because the historical phenotypic classification does not match the rRNA-based phylogeny (Vandamme et al., 1996). Historically, fermentative patterns were largely used to classify LAB and divide them into their various genera and species. This is particularly problematic for dairy Lactococcus lactis strains as they can carry multiple plasmids. The plasmid complement easily exceeds 150 kb, representing an important fraction of the L. lactis genome. Lactococcal plasmids specify many important metabolic functions such as lactose metabolism, citrate utilization, and peptide and amino acid uptake (Siezen et al., 2005). It is now, however, generally accepted that bacterial classification should reflect the natural, that is, phylogenetic, relationships between bacteria, and species are more often defined by percentage similarities between the 16S or 23S rRNA sequences (Woese, 1987). Despite the introduction of sequence identity criteria for species definition, the classification of genus and family can still be difficult to define, representing agglomerates of species and genera, respectively.

Most LAB belong to the phylum Firmicutes, class Bacilli, and order Lactobacillales. The newly available whole-genome sequences of various LAB have allowed the construction of phylogenetic trees with increased resolution and robustness (Makarova et al., 2006) (Fig. 1a). Maximum representation with parsimony (MRP) supertrees for LAB derived from 1160 single gene families have been generated (Kelly et al., 2010) and indicate similar relatedness to rpoA-based trees. Three closely related lineages can be defined: the Leuconostoc group, the Lactobacillus casei-Pediococcus group, and the Lactobacillus delbrueckii group. Further, the Pediococcus group is a sister to the Leuconostoc group within the Lactobacillus clade. The family Streptococcaceae, consisting of the Lactococcus and Streptococcus genera, forms a separate lineage and will be the focus of this review.

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Figure 1. Phylogenetic trees of LAB and Lactococcus species. (a) Rooted phylogenetic tree of sequenced LAB constructed on the basis of alignments of the DNA-dependent RNA polymerase. (b) Phylogenetic tree of Lactococcus species on the basis of complete 16S rRNA gene sequences. The minimum-evolution trees were built using mega 5.0 software. The species are colored according to the current taxonomy: Lactobacillaceae, blue; Leuconostocaceae, green; Streptococcaceae, red. Outgroup species are indicated in black. Based on Makarova et al. (2006).

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Despite the high sequence similarity between members of the family of Streptococcaceae, they inhabit a diverse range of environments. Members of the Lactococcus genus can be found in grasslands and are widely used in food fermentation, while the Streptococcus genus contains both health-promoting and disease-causing and tooth decay–causing species. While L. lactis is the best-known lactococcal species, six additional species have been added to this genus. These are Lactococcus garvieae, Lactococcus piscium, Lactococcus plantarum, Lactococcus raffinolactis, Lactococcus chungangensis, and Lactococcus fujiensis, which have been isolated from diverse habitats ranging from fish to activated sludge foam (Schleifer, 1987; Williams et al., 1990; Cho et al., 2008; Cai et al., 2011). Lactococcus lactis is further subdivided into four subspecies: L. lactis ssp. cremoris, L. lactis ssp. hordniae, L. lactis ssp. lactis, and the newly identified L. lactis ssp. tructae (Perez et al., 2011). Historically, L. lactis ssp. were referred to as lactic streptococci but were reassigned to their own genus in 1985 in order to distinguish them from the pathogenic streptococci (Schleifer et al., 1985; Schleifer, 1987). A phylogenetic tree based on 16S rRNA gene alignments of the Lactococcus species isolated thus far reiterates discrepancies between 16S rRNA gene sequence information and phenotypic characteristics of Lactococcus species (Fig. 1b). For example, the closely related L. lactis ssp. cremoris and L. lactis ssp. tructae were isolated from diverse environments – dairy products and intestinal mucous of trout, respectively – and have very different carbohydrate utilization patterns (Perez et al., 2011). The classification of L. lactis ssp. lactis and ssp. cremoris is especially problematic because of the occurrence of ‘cremoris type’ genotypes with ‘lactis type’ phenotypes. A good example for this is the type stain L. lactis ssp. cremoris MG1363. The reason for this is that while the 16S rRNA gene sequences of L. lactis ssp. lactis and ssp. cremoris differ by < 0.7% (Salama et al., 1991), they share only 85% identity at the genome level (Wegmann et al., 2007). This value is only slightly higher than that between Escherichia coli and Salmonella typhimurium (McClelland et al., 2001).

Like many other LAB, Streptococcus thermophilus is used in dairy starter cultures but the Streptococcus genus also contains members that are pathogenic to humans and other mammals. Streptococcus mutans and Streptococcus pneumoniae have been particularly well studied owing to their impact on human health. Streptococcus mutans is one of the major causative agents of dental caries (tooth decay) because of its rapid acidification of the environment caused by the end products of its sugar metabolism, while S. pneumoniae, also known as pneumococcus, can cause invasive diseases such as pneumonia and meningitis (Musher, 1992; Mufson, 1999; Mason et al., 2007).

The central metabolic pathways of the Streptococcaceae family have been extensively studied because of their relevance in the industrial use of some species (e.g. L. lactis), as well as their influence on virulence (e.g. S. pneumoniae and Streptococcus pyogenes). Recent developments in high-throughput proteomic and DNA-microarray techniques, in in vivo NMR studies, and importantly in whole-genome sequencing have resulted in new insights into the metabolism of the Streptococcaceae family. In this review, we will discuss the central metabolic pathways and their regulation, including the heme-dependent aerobic electron transport chain (ETC) and the ATP-generating arginine deiminase (ADI) pathway, present in the Streptococcaceae. Special emphasis will be on the adaptation of metabolic pathways of the sequenced strains to the nutrient-rich environments they inhabit.

Central metabolism

  1. Top of page
  2. Abstract
  3. Introduction
  4. Central metabolism
  5. The effect of oxygen on metabolism of Streptococcaceae
  6. Proton motive force generation by the F1Fo ATPase and solute transport
  7. ADI pathway
  8. Evidence from genome sequences for adaptation of Streptococcaceae to nutrient-rich environments
  9. Future perspectives
  10. References

Metabolic energy can be defined as the free energy stored in the ATP pool and in the electrochemical protein gradient across the cytoplasmic membrane. Lactococci and streptococci are classified as facultative anaerobes, unable to fully oxidize sugars to CO2 via the TCA cycle, even when oxygen is present. Under anaerobic conditions, there is no net production of reducing equivalents and ATP is generated by substrate-level phosphorylation. These bacteria convert a large fraction of the hexose sugar substrate to pyruvate via glycolysis and then to lactate in the redox balance step. Alternatively, pyruvate is converted to the mixed-acid products formate, ethanol, acetate, 2, 3-butanediol, and acetoin, generating extra ATP (Kandler, 1983). In a detailed study on L. lactis ssp. lactis NCDO 2118 using 14C-labeled glucose and amino acids, it was observed that 91% of the fermentation end products were derived from glucose, while the other 9% were derived from serine via pyruvate (Novak & Loubiere, 2000). Relative to the carbon flux from hexose sugars to lactate, the calculated carbon flux to biomass through the pathways branching from glycolysis was small: 5% with growth on minimal medium (Novak & Loubiere, 2000). In addition to glucose, glutamate-derived products formed a large fraction of the biomass. The study reiterated the current notion that glucose is only a catabolic substrate.

Sugar import

Most hexose sugars and disaccharides used by lactococci and streptococci are taken up by phosphotransferase systems (PTSs) but non-PTS permeases are also present (Fig. 2). For example, in L. lactis glucose can be imported via the mannose or cellobiose PTSs or the glucose permease GlcU (Castro et al., 2009). In S. mutans alone, PTS systems have been identified for glucose (Schachtele & Mayo, 1973), mannitol, glucitol (Brown & Wittenberger, 1973; Maryanski & Wittenberger, 1975), lactose (Calmes, 1978), sucrose (Slee & Tanzer, 1979; St Martin & Wittenberger, 1979), mannose (Vadeboncoeur, 1984; Néron & Vadeboncoeur, 1987), fructose (Gauthier et al., 1984ab), maltose (Würsch & Koellreutter, 1985), and trehalose (Poy & Jacobson, 1990).

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Figure 2. Hexose and disaccharide sugar utilization in the family of Streptococcaceae. Glucose is either internalized and concomitantly phosphorylated the PTS systems (PtcABC or PtnABCD) or imported by the GlcU permease and phosphorylated by glucose kinase. PTS systems are represented by circles, while non-PTS sugar import systems are represented by squares. Glucose-6-phosphate (G6P) is then converted to fructose-6-phosphate (F6P) by phosphoglucose isomerase (PGI) and subsequently to fructose-1,6-bisphosphate (FBP) by phosphofructokinase (PFK). FBP is converted by fructose-bisphosphate aldolase (FBA) to dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP), two products that can be interconverted by triosephosphate isomerase (TPI). GAP is in turn converted to pyruvate via 1,3PG, 3PG, 2PG, and phosphoenolpyruvate (PEP), reactions that are catalyzed by GAP dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), phosphoglycerate mutase (PMG), enolase (ENO), and pyruvate kinase (PK), respectively. PEP can also be converted to pyruvate via the sugar PTSs. In the homolactic fermentation pathway, pyruvate is converted to lactate by lactate dehydrogenase (LDH), which uses the reducing equivalents generated in glycolysis. In the mixed-acid fermentation pathway, pyruvate is converted to acetyl-CoA by pyruvate formate lyase (PFL) (under anaerobic conditions) producing formate or by pyruvate dehydrogenase (PDH) (under aerobic conditions). Acetyl-CoA is subsequently converted to ethanol and acetate by acetaldehyde-CoA/alcohol dehydrogenase (ADHE) and alcohol dehydrogenase (ADHA), and phosphotransacetylase (PTA) and acetate kinase (ACKA), respectively. The formation of acetate from acetyl-P generates ATP. When pyruvate levels are high, acetoin and 2,3-butanediol are formed (Snoep et al., 1992) through reactions that are catalyzed by acetolactate synthase (ALS), alpha-acetolactate decarboxylase (ALDB), acetoin reductase (BUTA), and 2,3-butanediol dehydrogenase (BUTB). When galactose or lactose is utilized, each of these sugars is fed into glycolysis by either the tagatose or Leloir pathways. In the tagatose pathway, lactose is imported by the lactose PTS system (LacEF) and the lactose-6-phosphate (Lac6P) formed is hydrolyzed internally by phospho-β-galactosidase (Pβgal) into glucose and galactose-6-phosphate (Gal6P). Galactose is also imported by the lactose PTS system, forming galactose-6-phosphate. The glucose (from lactose) is metabolized to pyruvate via glycolysis, while Gal6P is converted to tagatose-6-phosphate (T6P) by galactose-1-phosphate (Gal1P) and subsequently to FBP by tagatose-phosphate kinase (TPK). FBP is converted by tagatose-bis-phosphate aldolase (TBA) to DHAP and GAP, which can then be fed into glycolysis. All genes involved in the lactose pathway, lacABCDFEG, are contained in one operon that is often found on plasmids (de Vos & Vaughan, 1994). In the Leloir pathway, lactose is imported by the lactose permease, LacS, after which it is hydrolyzed into glucose and α-galactose by converted β-galactosidase. Galactose is taken up by the galactose permease (GalP) and converted to α-galactose by galactokinase (GalM). The α-galactose is phosphorylated by galactokinase (GalK) to form α-galactose-1-phosphate (α-Gal1P) from which α-glucose-1-phosphate is formed by galactose-1-phosphate uridylyltransferase/UDP-glucose 4-epimerase (GalT/GalE). From this, glucose-6-phosphate can then be formed by phosphoglucomutase (PGM). Sucrose is imported via the sucrose-specific PTS, ScrA, or via PttB, a trehalose-specific PTS with low affinity for sucrose. Sucrose can also be imported by MSM (multiple sugar metabolism) systems, which are ABC-type transporters in Streptococcus species (Russell et al., 1992) (not illustrated). PTS-imported and phosphorylated sucrose-6-phosphate (Suc6P) is hydrolyzed by sucrose-6-phosphate hydrolase (S6PH) to G6P and fructose. The fructose is subsequently phosphorylated by fructokinase. Maltose is also imported by an ABC-type transporter, MalEFG, and maltose is degraded to β-glucose-1-phosphate (β-G1P) by maltose phosphorylase (MP), which is converted in a reversible reaction to G6P by β-phosphoglucomutase (β-PGM). The highlighted arrows indicate reactions in which the NAD+ needed for ATP synthesis during glycolysis is regenerated. Nomenclature used is for Lactococcus lactis.

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When phosphorylated at His-15 by the PEP and enzyme I, the HPr protein acts as a phosphocarrier for sugar PTSs (Fig. 3). HPr-His-P can also function in the regulation of carbohydrate metabolism. HPr-His-P can phosphorylate histidyl resides in non-PTS proteins such as glycerol kinase (Charrier et al., 1997) and non-PTS transporters that contain an EIIAGlc domain (Gunnewijk et al., 1999), stimulating their activity. HPr can be phosphorylated at Ser-46 by the metabolite-controlled HPr kinase/P-Ser-HPr phosphatase. HPr-Ser-P protein plays a role in catabolite repression (Deutscher et al., 1995; Luesink et al., 1999). HPr-Ser-P has also been implicated in inducer exclusion (Saier, 1989; Saier et al., 1996; Titgemeyer & Hillen, 2002), and for Streptococcus salivarius, it has been shown that sugar uptake is relieved from glucose inhibition in HPr mutants or mutants unable to form HPr-Ser-P and glucose is no longer preferentially taken up and metabolized over lactose in the mutants (Gauthier et al., 1997; Monedero et al., 2001ab). This mode of regulation is not observed in S. mutans but is present in other streptococci such as S. pyogenes (Reizer & Panos, 1980; Reizer et al., 1983). It was previously thought that HPr-Ser-P was directly involved in inducer expulsion. In inducer expulsion, the cell rapidly ejects the nonpreferred carbon sources when exposed to easily metabolized ones, most often glucose. This applies to sugars internalized by PTS. The accumulated sugar-P is dephosphorylated by a sugar-P phosphohydrolase and then expelled into the external medium (Thompson & Saier, 1981; Reizer et al., 1983; Reizer & Saier, 1983; Viana et al., 2000). Recent experiments, however, have shown that HPr-Ser-P does not function in inducer expulsion in L. lactis and Lactobacillus casei as mutants unable to form HPr-Ser-P expelled nonmetabolizable methyl β-d-thiogalactoside (TMG) (Monedero et al., 2001ab; Dossonnet et al., 2000).

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Figure 3. Hpr- and CcpA-mediated catabolite repression. During carbohydrate uptake and phosphorylation, HPr becomes phosphorylated at His-15 by PEP and enzyme I of the PTS. HPr-His-P acts as a phosphocarrier transferring its phosphoryl group to a sugar-specific EIIA. The phosphoryl group is in turn donated to the corresponding EIIB, after which it is finally transferred to the carbohydrate bound to EIIC. When HPr is phosphorylated at Ser-46 by HPr kinase/HPr-Ser-P phosphatase, it binds to the transcriptional regulator CcpA. CcpA complexed with the HPr-Ser-P binds to certain cre sites ensuring hierarchal and efficient use of preferred sugars.

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In addition, HPr acts as an allosteric effector to regulate transcription through the catabolite control protein CcpA. CcpA is a transcriptional regulator that binds to cis-elements designated cre sites (catabolite response elements) and can act as both an activator and a repressor of gene expression (Luesink et al., 1998). CcpA binds certain cre sites, such as those upstream of catabolic operons involved in galactose and maltose utilization, together with the serine-phosphorylated form of HPr, thus ensuring hierarchal and efficient use of preferred sugars (Titgemeyer & Hillen, 2002; de Vos & Vaughan, 1994; Luesink et al., 1998; Deutscher et al., 1995) (Fig. 3). In L. lactis ssp. cremoris, the link between either gene repression or activation by CcpA and the location of the cre site has been investigated (Zomer et al., 2007). Gene activation was observed when cre sites were located upstream of the −35 sequence, while repression was observed when the cre site was in or downstream of the putative −35 and −10 sequences.

Glycolysis

During glycolysis, ATP is generated, a reaction that requires NAD+. Glucose imported by PTSs is concomitantly phosphorylated and fed into glycolysis. Other hexose sugars or disaccharides must first be converted and/or hydrolyzed to sugar phosphate species that can be glycolysed (Fig. 2). The conversion of lactose and sucrose into glycolytic intermediates has been extensively studied in L. lactis and S. mutans, respectively. Streptococcus mutans is the major cause of dental caries owing to its formation of the weak acid lactate from ingested sucrose (Munro et al., 1991). Although this bacterium can hydrolyze sucrose extracellularly by secreting fructosyl- and glucosyltransferases (Newbrun, 1983), the majority of dietary sucrose is metabolized intracellularly (Tanzer et al., 1972). Sucrose is imported and phosphorylated via either the dedicated sucrose PTS or the trehalose PTS (Poy & Jacobson, 1990) after which phosphorylated sucrose is hydrolyzed to glucose-6-phosphate (G6P) and fructose, the latter of which is subsequently phosphorylated by fructokinase to fructose-6-phosphate (F6P) (Postma et al., 1993; Chassy & Porter, 1979). The sugar phosphate compounds can then be channeled into the glycolytic pathway. Galactose and lactose are converted to glycolytic intermediates in L. lactis ssp. via either the tagatose or Leloir pathways (summarized in Fig. 2). The main difference between the two pathways is the manner in which the sugars are imported. In the tagatose pathway, lactose and galactose are imported and concomitantly phosphorylated via the PTSlac (Van Rooijen et al., 1991), while in the Leloir pathway the sugars are imported by dedicated permeases (Grossiord et al., 2003; Thomas et al., 1980; Thompson, 1980). This influences the products formed, with the tagatose pathway forming G6P and the trioses, DHAP and GAP, and the Leloir pathway producing G6P and glucose. The use of the Leloir and/or the tagatose pathway for galactose utilization is strain dependent (Thomas et al., 1980).

Control of glycolytic flux

No single enzymatic step controls the rate of glycolysis in L. lactis, and control appears to be distributed over many glycolytic steps, sugar uptake, and lactate efflux (Koebmann et al., 2002). This is supported by experiments that modulated glyceraldehyde-3-phosphate dehydrogenase (GAPDH), l-lactate dehydrogenase (LDH), phosphofructokinase (PFK), or pyruvate kinase (PK) expression individually. Decreasing but not increasing GAPDH expression had an effect on the glycolytic flux (Solem et al., 2003), while if the expression levels of LDH, PFK, and PK were increased or decreased together, the flux through glycolysis decreased (Koebmann et al., 2002). Another study showed that decreasing PFK levels alone in L. lactis did reduce the glycolytic flux and growth rate (Andersen et al., 2001). The reason for this discrepancy may be that different strains and/or methods were used.

The distribution of flux control over a number of enzymatic steps is partly regulated by CcpA. In addition to controlling the transcription of sugar-specific operons, CcpA acts as an activator of transcription of the las operon genes pfk, pyk, and ldh, encoding PFK, PK, and LDH, by binding to a cre site upstream of the operon (Luesink et al., 1998; Karlin et al., 2004). CcpA and the metabolite fructose-1, 6-bisphosphate (FBP) mediate both transcriptional and allosteric activation of LDH to ensure the rapid formation of lactate. In Bacillus subtilis, FBP has been shown to enhance the interaction of HPr-Ser-P with CcpA and the interaction of the complex with cre sites (Seidel et al., 2005; Schumacher et al., 2007). In addition, FBP acts as allosteric regulator of LDH, increasing its activity (Hillier & Jago, 1982). High levels of FBP result in the activation of pyk by CcpA in turn decreasing the amount of PEP, thus fine-tuning PTS sugar transport. Regulation by intracellular FBP levels applies to high glycolytic fluxes. When the glycolytic flux is low, the flux is controlled by the ATP demands of the cell (Koebmann et al., 2002).

Homolactic and mixed-acid fermentation

The pyruvate generated by glycolysis can have a number of fates in streptococci and lactococci. In general, under anaerobic conditions and when easily utilizable sugars, that is, glucose, are in excess, pyruvate is converted solely to lactate by LDH. Alternatively, pyruvate can be channeled through the pyruvate formate lyase (PFL) pathway forming formate, ethanol, and acetate. Under aerobic conditions, PFL is inhibited and is replaced by pyruvate dehydrogenase (PDH) in lactococcal and some streptococcal species or pyruvate oxidase (PO) in other streptococci (Taniai et al., 2008; Melchiorsen et al., 2000). The oxygen sensitivity of these enzymes is important in dental health as they allow streptococci such as Streptococcus gordonii, Streptococcus mitis, Streptococcus oralis, and Streptococcus sanguis to compete successfully with other bacteria at sites of the oral cavity that are freely exposed to saliva, while S. mutans is restricted to anaerobic sites such as those in between teeth (Abbe et al., 1991). The mixed-acid fermentation pathway in L. lactis has an important influence on the flavor of dairy products as the short-chain compounds diacetyl, acetoin, acetate, acetaldehyde, and ethanol all have an effect on the palatability (Cogan & Hill, 1993; Escamilla-Hurtado et al., 1996; Henriksen & Nilsson, 2001; Syu, 2001). Diacetyl and acetoin in particular are major flavor compounds in cheeses with creamy buttery aromas such as cheddar and gouda (Dacremont & Vickers, 1994; Chritstensen & Reineccius, 1995; Curioni & Bosset, 2002). In contrast to other members of the Streptococcaceae family, S. thermophilus only utilizes the homolactic fermentation pathway owing to the presence of pseudogenes in some primary metabolic pathways (Pastink et al., 2009).

Switch from homolactic to mixed-acid fermentation

It has been proposed that a rapid flux through glycolysis is likely to result in homolactic fermentation, while diminished rates of sugar metabolism provoke a shift toward mixed-acid fermentation. For example, when the glycolytic flux is low because of inefficient sugar uptake or lack of tagatose pathway activity, some benefit can be derived from switching to mixed-acid fermentation as it generates more ATP than the homolactic pathway, even though the end products of the mixed-acid pathway are more inhibitory than lactate to the bacterium itself (Loubiere et al., 1997). In L. lactis ssp. lactis strain IL1403, the rapid uptake and concomitant phosphorylation of fructose, mannose, glucose, or sucrose by efficient PTS systems supports rapid rates of sugar metabolism and therefore homolactic fermentation (Bolotin et al., 2001; Cocaign-Bousquet et al., 1996). In contrast, mixed-acid fermentation is observed for growth on sugars such as galactose, gluconate, xylose, maltose, or ribose, which are not imported by a PTS system. In L. lactis strains, such as L. lactis ssp. cremoris NCDO 712, which contain a plasmid encoding a lactose-specific PTS system, lactose fermentation is homolactic (Gasson, 1983).

Expression levels of genes of the enzymes involved in fermentation alone do not explain the shift between homolactic and mixed-acid fermentation. Triose phosphates have a controlling effect on PFL activity, and during glucose fermentation, when triose phosphate pools are high, the activity of this enzyme would be completely inhibited (Thomas et al., 1980; Takahashi et al., 1982; Garrigues et al., 1997). Also, the activities of LDH and GAPDH can be modulated in an opposing but similar manner by the NADH/NAD+ ratio in the cell resulting in a coordinated effect on flux through the glycolytic and fermentation pathways (Garrigues et al., 1997). The activity of LDH is inhibited by increasing concentrations of NAD+, while the activity of GAPDH is inhibited by increasing concentrations of NADH. Artificial changing of the NADH/NAD+ ratio in L. lactis can redirect fermentation from the homolactic to the mixed-acid type with acetoin and diacetyl produced (De Felipe et al., 1998). Whole-genome sequencing has revealed the presence of at least five NADH dehydrogenase/oxidase genes in the genomes of L. lactis ssp. lactis strains IL1403 and KF147, L. lactis ssp. cremoris strain SK11 and four in the genome of L. lactis ssp. cremoris strain MG1363. Each of these may affect the mode of fermentation.

The fermentation mode can be affected by oxygen availability, and in the case of oral streptococci, this has an important effect on the formation of dental caries. In the deep layers of dental plaque, the supply of sugar is limited, the pH is frequently below 7, and these sites are highly anaerobic (Kenney & Ash, 1969). These conditions favor streptococcal PFLs because the intracellular levels of glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP), which inhibit PFL, are low and the PFLs are prevented from irreversible inactivation by oxygen. Also, S. mutans has a PFL-activating system that operates optimally at pH 6.8. This system includes a PFL-activating enzyme that itself is activated by an electron transport system that transfers an electron from NADH to the PFL-activating enzyme (Takahashi-Abbe et al., 2003). The resulting rapid acid production in the deep layers of dental plaque likely confers the high cariogenic potential of this microorganism. This would be different to the situation in immature thin dental plaque and superficial layers of dental plaque where there would be transient infiltration of oxygen.

The effect of oxygen on metabolism of Streptococcaceae

  1. Top of page
  2. Abstract
  3. Introduction
  4. Central metabolism
  5. The effect of oxygen on metabolism of Streptococcaceae
  6. Proton motive force generation by the F1Fo ATPase and solute transport
  7. ADI pathway
  8. Evidence from genome sequences for adaptation of Streptococcaceae to nutrient-rich environments
  9. Future perspectives
  10. References

While members of the family Streptococcaceae are most often characterized by their fermentative behavior under anaerobic growth conditions, these bacteria are aerotolerant. Lactococcus lactis ssp. and Streptococcus species are defined as catalase negative but the presence of the flavoproteins NADH oxidase and NADH peroxidase and a manganese-containing superoxide dismutase allows them to grow under aerobic conditions (Hansson & Häggström, 1984; Sanders et al., 1995).

Despite the presence of these enzymes, exposure to oxygen does result in cell damage because of the formation of reactive oxygen species (Anders et al., 1970; Duwat et al., 1995; Miyoshi et al., 2003). The damaging effects of oxygen are, however, markedly reduced when L. lactis is grown in the presence of heme (Sijpesteijn, 1970; Mickelson, 1972; Duwat et al., 2001). This is likely due to a shift from homolactic to mixed-acid fermentation, more complete glucose utilization, and energy generation by NADH oxidation via an ETC. (Duwat et al., 2001). The observation that functional cytochromes are synthesized in heme-containing growth medium was made over 40 years ago (Whittenbury, 1964; Bryan-Jones & Whittenbury, 1969; Sijpesteijn, 1970; Ritchey & Seeley, 1974), the genetic basis for which is evident in the whole-genome sequences published thus far. Lactococcus lactis strains all contain genes for menaquinone synthesis (men operon), the late steps in heme biosynthesis (hemH, hemK, hemN) as well as those for a membrane-bound NADH dehydrogenase (noxA, noxB) and cytochrome d oxidase and its synthesis (cytABCD) (Bolotin et al., 2001; Brooijmans et al., 2007; Wegmann et al., 2007) (illustrated in Fig. 4). Furthermore, a clear build-up of a ΔΨ has been demonstrated when heme was added to aerated cultures showing that a functional ETC. is indeed formed in L. lactis cells (Brooijmans et al., 2007).

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Figure 4. Oxygen-related metabolism in Streptococcaceae. In the absence of heme, Streptococcaceae are able to reduce O2 to H2O and regenerate NAD+ via the NADH oxidase, NoxE, together with the alkaline hydroperoxide system (AhpR). Alternatively, in Lactococcus lactis, the presence of exogenous heme allows for the synthesis of cytochromes and hence the generation of a PMF via an ETC. The ETC is formed by the membrane-bound NADH dehydrogenase complex (NoxAB) and the cytochrome d oxidase (CydAB). ATP is generated from the PMF built up by the ETC via the F1Fo ATPase. When oxygen is present, pyruvate formate lyase is inactivated and is replaced by pyruvate dehydrogenase (PDH), an enzyme from the mixed-acid pathway. Ethanol is produced from pyruvate via the action of acetaldehyde-CoA/alcohol dehydrogenase (ADHE) and alcohol dehydrogenase (ADHA). Pyruvate can also be converted to acetate via the oxygen-dependent enzyme pyruvate oxidase (PO) and acetate kinase (ACKA). Through the action of lactate oxidase (LO), acetate can be formed from pyruvate, allowing for the production of additional ATP via the mixed-acid pathway.

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The import of heme is likely performed by the ABC-type iron uptake system encoded by the fhu operon (Gaudu et al., 2003). CcpA is important in regulating the expression of these genes in the presence of oxygen. Expression of fhuR, which encodes a repressor of the heme transport system genes fhuB, fhuG, and fhuD, is activated by CcpA, and in this way, CcpA coordinates oxygen, carbon, and iron metabolism (Gaudu et al., 2003).

The original habitat of L. lactis, the plant surface or phyllosphere (Kelly et al., 2010), may contain heme but the actual source is unclear. In the absence of heme, such as is the case in milk, the reduction of O2 to H2O is likely due to the NADH oxidase encoded by noxE (Jeanson et al., 2009; De Felipe & Hugenholtz, 1999), which is only active in the presence of oxygen. NoxE has been shown to play an important role in the regeneration of NAD+ during aerobic metabolism in L. lactis and S. mutans (Higuchi et al., 1993; De Felipe et al., 1997). These bacteria are also able to reduce O2 to H2O via the alkyl hydroperoxide reductase (AhpR) composed of the H2O2-producing NADH oxidase AhpF and a peroxyredoxine (AhpC), which reduces H2O2 to H2O (Higuchi et al., 1999; Tachon et al., 2010). Genes encoding for thiol peroxidase (tpx) and glutathione reductase (gshR) have been identified in L. lactis and may also be involved in responses of this bacterium to aerobic growth conditions. Notably CcpA is thought to play a role in regulating noxE expression in response to oxygen levels (Jensen et al., 2001). Increased NADH oxidase activity would eliminate intracellular oxygen and decrease the NADH pool, which may influence the shift toward mixed-acid fermentation observed in aerobically grown cells (Gaudu et al., 2003).

Streptococcus agalactiae, also known as group B Streptococcus (GBS), is able to respire when heme and menaquinones are supplied (Yamamoto et al., 2005). Respiration is an important virulence factor in S. agalactiae, allowing persistence in the blood stream and therefore dissemination of the streptococcal infection (Yamamoto et al., 2005). The heme may be acquired from degraded red blood cells, while the source of menaquinones may be other bacteria present in the host. Respiring L. lactis has been shown to provide S. agalactiae with the menaquinones necessary for respiration (Rezaïki et al., 2008). Not all streptococci are capable of respiration. For example, S. mutans lacks cytochromes, heme-containing proteins, and catalases but is able to grow in the presence of oxygen, and all genes known to be involved in oxidative stress are present (Yamamoto et al., 2000; Ajdic et al., 2002).

Gene regulation in response to oxygen plays an important role in the virulence of many species of Streptococcus, and the production of H2O2 gives them a competitive advantage in their specific niches. This phenomenon has been well studied in S. pneumoniae that contains, in addition to the PFL and PK enzymes, the H2O2-forming flavoprotein PO. When oxygen is present, pyruvate can be converted to acetate via acetyl phosphate, yielding ATP (Spellerberg et al., 1996) (Fig. 4). Acetyl phosphate is formed by PO, accounting for most of the H2O2 produced by aerobically growing S. pneumoniae cells (Spellerberg et al., 1996). The production of H2O2 has an inhibitory effect on other inhabitants of the upper respiratory tract, such as Haemophilus influenzae, Moraxella catarrhalis, and Neisseria meningitidis (Pericone et al., 2000), and contributes to the virulence of S. pneumoniae (Regev-Yochay et al., 2007). The extra ATP generated by this pathway prolongs the survival of S. pneumoniae cells exposed to exogenously added H2O2 (Pericone et al., 2003). Although H2O2 production is lethal to S. pneumoniae, causing the stationary-phase apoptosis observed during aerobic growth, it confers a colonization advantage to this bacterium in the upper respiratory tract. This would explain the persistence of such apparently suicidal genes (Regev-Yochay et al., 2007). A novel gene, poxL, encoding PO has also been identified in L. lactis strains IL1403, MG1363, and KF147 (Bolotin et al., 2001; Wegmann et al., 2007; Siezen et al., 2010). Although this enzyme has not been studied in L. lactis, PO activity has been detected under aerobic growth conditions in the LAB Lb. plantarum (Goffin et al., 2006).

Streptococcus pneumoniae possesses another H2O2-forming flavoprotein, l-lactate oxidase (LO). This enzyme was first described in S. pneumoniae in 1959 (Udaka et al., 1959) and catalyzes the formation of pyruvate and H2O2 from lactate and O2. LO is also found in S. pyogenes (Seki et al., 2004). The two H2O2-forming flavoproteins can act in a concerted manner under aerobic conditions to obtain a greater amount of energy from glucose than under anaerobic fermentative conditions. H2O2 formation in S. pneumoniae is independent of the glucose concentration, while in S. pyogenes there is no H2O2 build-up or LO activity until the glucose in the medium is exhausted (Gibson et al., 2000). This is also true for the fish pathogen Streptococcus iniae (Gibello et al., 1999). The constitutive expression of the spx (PO) and lox (LO) genes in S. pneumoniae may therefore be unique to this bacterium (Taniai et al., 2008). Still unclear, however, is the achievement of redox balance in the pathways just described. The recycling of NADH is apparently incomplete because only part of the pyruvate derived from glucose is available for NADH oxidation by LDH. It has been suggested that cycling between pyruvate and lactate occurs, in which a portion of the pyruvate pool produced by LO is reduced back to lactate by LDH with concomitant oxidation of NADH. Alternatively, an NADH oxidase could perform the necessary regeneration of NAD+ (Auzat et al., 1999).

Proton motive force generation by the F1Fo ATPase and solute transport

  1. Top of page
  2. Abstract
  3. Introduction
  4. Central metabolism
  5. The effect of oxygen on metabolism of Streptococcaceae
  6. Proton motive force generation by the F1Fo ATPase and solute transport
  7. ADI pathway
  8. Evidence from genome sequences for adaptation of Streptococcaceae to nutrient-rich environments
  9. Future perspectives
  10. References

While many anaerobic bacteria utilize sugars in the ways described above, lactococci and streptococci are unique in their avoidance of de novo synthesis of many macromolecular building blocks, especially amino acids (Juillard et al., 1995; Poolman & Konings, 1988). These bacteria therefore make great nutritional demands on the growth medium, which are accommodated by a wide variety of substrate transporters (Poolman, 1993).

Nutrient uptake is an energy-expensive process. Although L. lactis has a relatively small genome, analysis of the sequenced genomes reveals that between 8.5% and 10% of the predicted proteins are transporter proteins. This is comparable to E. coli, although L. lactis strains have a higher proportion of ABC transporters relative to secondary transporters (Konings, 2002). Although energetically costly, ABC transporters usually have very high affinities for their solutes and catalyze transport at high rates. This may allow L. lactis to efficiently scavenge essential solutes from the environment, giving it a competitive advantage (Konings, 2002). Transport via secondary transporters, that is, those coupled to the influx of protons driven by the proton motive force (PMF), is less energetically expensive than uptake via ABC transporters. The main proton pump in L. lactis and Streptococcus species responsible for PMF generation is the F1Fo ATPase, which pumps protons at the expense of metabolic ATP (Maloney, 1982). As already mentioned, under aerobic conditions with a heme source, the ETC. also leads to the generation of a PMF (Brooijmans et al., 2007).

A more energy-efficient manner of nutrient uptake than the ones described above is by symport or antiport in which the passage of compounds across the membrane is coupled resulting in the export of one proton. In S. mutans, malate fermentation can be coupled to ATP synthesis (Sheng & Marquis, 2007). In this processes, known as malolactic fermentation, dicarboxylic l-malate is converted to monocarboxylic l-lactate and carbon dioxide. The uptake of malate is coupled to the export of lactate, resulting in the transport of one proton. The PMF built up in this way can subsequently be used for ATP synthesis by F1Fo ATPase (Poolman et al., 1991). Antiport can also make the cost of transport neutral as, for example, in the case of the arginine/citrulline transporter. Thus, ATP can be generated via the arginine deiminase pathway (discussed in detail below) rather than being used up for arginine transport into the cell.

Although lactate is the main fermentation product of LAB, the manner in which it leaves the cell has not been elucidated for all LAB species. Lactate is a weak acid with a pKa of 3.87, and at an intracellular pH of 7, almost all of it is dissociated and therefore membrane-impermeable. Biochemical studies in L. lactis have provided evidence for lactate symport with two or three protons (Michels et al., 1979; Otto et al., 1980ab; ten Brink et al., 1985), although the protein(s) responsible for this transport have yet to be identified. The transport of lactate together with protons could therefore generate 0.66–0.5 mol ATP per mol lactate transported. If the continuous production of large quantities of metabolic end products was coupled to protons, it would contribute significantly to the PMF and overall production and conservation of energy (Michels et al., 1979; ten Brink & Konings, 1982). This prediction is supported by the observation that L. lactis has an increased biomass yield when co-cultured with Pseudomonas stutzeri, which removes the lactate from the medium (Otto et al., 1980ab).

More important than for the uptake of nutrients, proton translocation is used in the maintenance of a proper internal pH in streptococcal and lactococcal species (Harold, 1986). The internal pH is not fully maintained in these bacteria, and acidification of the cytoplasm does occur when the pH of the external medium decreases. In vivo NMR studies in L. lactis to measure the internal pH revealed that while the cytoplasmic pH is 7.2 at an external pH of 6.5, it drops to 6 when the external pH is 4.8 (Neves et al., 2002ab). While the F1Fo ATPase can generate ATP under physiological conditions (Maloney, 1982), it acts predominantly as a proton pump (Konings et al., 1989) and the acid tolerance of LAB is based primarily on this activity (Quivey et al., 2000, 2001; Bender et al., 1986; Suzuki et al., 2000). Ammonium, which is produced during the degradation of arginine, may also function as a neutralizing agent against protons (see ADI pathway below). The enzymes responsible for the production of ammonium are extremely acid tolerant, and in Streptococcus rattus and S. sanguis, the enzymes have been shown to function at pH values as low as 3 (Casiano-Colón & Marquis, 1988).

ADI pathway

  1. Top of page
  2. Abstract
  3. Introduction
  4. Central metabolism
  5. The effect of oxygen on metabolism of Streptococcaceae
  6. Proton motive force generation by the F1Fo ATPase and solute transport
  7. ADI pathway
  8. Evidence from genome sequences for adaptation of Streptococcaceae to nutrient-rich environments
  9. Future perspectives
  10. References

The ADI pathway, through which arginine is degraded into ammonium and carbon dioxide with concomitant production of ATP, is widespread in Streptococcaceae (Abdelal, 1979; Cunin et al., 1986). The ADI pathway in Streptococcus species is commonly referred to as the arginine deiminase system (ADS) (Quivey et al., 2001).

The ADI pathway consists of the three enzymes, arginine deiminase (ADI), catabolic ornithine carbamoyltransferase (OTC), and carbamate kinase (CK) (Cunin et al., 1986) (Fig. 5a). ADI is encoded by genes designated as either arcA (for arginine catabolism) or sagP (for streptococcal acid glycoprotein) in some streptococcal species. The enzyme hydrolyzes arginine into citrulline and ammonium. The presence of ADI activity was used historically to differentiate between the L. lactis ssp. lactis and cremoris with L. lactis ssp. lactis strains all containing active ADI enzymes (Crow & Thomas, 1982). Lactococcus lactis ssp. cremoris strain MG1363 is atypical in that it possesses a fully functional ADI pathway. The product of ADI, citrulline, is metabolized by catabolic ornithine carbamoyltransferase or ornithine transcarbamoylase (OTC) into ornithine and carbamoyl phosphate. Ornithine, a byproduct, is excreted out of the cell via the arginine–ornithine antiporter (Poolman et al., 1987). Electroneutral exchange of equimolar amounts of arginine and ornithine takes place without the need for an external energy supply. OTC genes are often misannotated; for example S. mutans arcB and L. lactis argF2 are in fact putrescine carbamoyltransferase genes (Naumoff et al., 2004). CK, the product of the arcC gene, catalyzes the final step in arginine catabolism, the conversion of carbamoyl phosphate into carbon dioxide and ammonium with concomitant transfer of the high-energy phosphate group to ADP, generating ATP. Carbamoyl phosphate also serves as a substrate for de novo pyrimidine biosynthesis. The arcC gene is duplicated in many species, either residing in tandem, as in L. lactis ssp. lactis IL1403 and L. lactis ssp. cremoris MG1363, or at distant loci on the chromosome as in L. lactis ssp. lactis KF147.

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Figure 5. Arginine deiminase pathway in Streptococcaceae. (a) Arginine is imported into the cell via ArcD antiporter in equimolar amounts in exchange for ornithine, which is a waste product of arginine catabolism. In the cytoplasm, arginine is degraded into citrulline and ammonia by the arginine deiminase enzyme (ADI) enzyme. Catabolic ornithine transcarbamoylase (OTC) catalyzes the conversion of citrulline into ornithine and carbamoyl phosphate. Carbamate kinase (CK) then degrades citrulline to carbon dioxide and ammonium with the concomitant production of ATP. Carbamoyl phosphate can also be used as a precursor for de nova synthesis of pyrimidines. (b) Organization of ADI pathway gene cluster in Lactococcus lactis IL1403.

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Besides these genes coding for metabolic enzymes, transcription factor genes such as ArgR, AhrC, and ArcR are also frequently found in the ADI pathway gene cluster, for example, in L. lactis, S. pyogenes, and S. pneumoniae, as is the gene argS encoding arginyl-tRNA synthetase (Fig. 5b). The arginine–ornithine antiporter gene arcD is also frequently found in the ADI pathway gene cluster. In L. lactis MG1363, ArgR functions as a DNA-binding protein repressing the expression of the ADI pathway gene cluster. It has been proposed that ArgR binds DNA in a homohexameric form. AhrC (ArgR homologous regulator C), on the other hand, is an arginine sensor lacking DNA-binding ability. It functions as a cofactor relieving repression by ArgR through the formation of heterohexamers with ArgR (Larsen et al., 2005). The promoter regions of the ADI pathway gene cluster of L. lactis and S. gordonii contain a cre site, likely responsible for the repression of these genes by glucose via CcpA (Poolman et al., 1987; Dong et al., 2004). The ADI pathway gene cluster in S. gordonii contains two extra genes, namely flp and queA. The flp gene encodes for an Fnr-like protein and has been shown to be a transcriptional activator of the gene cluster (Dong et al., 2004). QueA is predicted to be an S-adenosylmethionine:tRNA ribosyltransferase isomerase responsible for the queosine modification of tRNAs. QueA has a role in the modulation of gene expression by exerting a negative effect on the translation of other ADI pathway transcriptional regulators (Liu et al., 2008). Although deletion of either the queA or ccpA genes had a measurable effect on arcA transcription, no changes in ADI enzyme activity were observed (Liu et al., 2008). This suggests that there is tight post-translational control over the amount of ADI pathway enzymes in the cell.

The ADI pathway produces ATP and arginine can support growth in L. lactis ssp. lactis strains as well as in another LAB, Enterococcus faecalis (Niven et al., 1941; Deibel, 1964; Simon et al., 1982). Typical L. lactis ssp. cremoris strains do not possess the ADI pathway but the effect hereof on pH tolerance and/or ATP production has not been studied.

Evidence from genome sequences for adaptation of Streptococcaceae to nutrient-rich environments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Central metabolism
  5. The effect of oxygen on metabolism of Streptococcaceae
  6. Proton motive force generation by the F1Fo ATPase and solute transport
  7. ADI pathway
  8. Evidence from genome sequences for adaptation of Streptococcaceae to nutrient-rich environments
  9. Future perspectives
  10. References

The genomes of members of the family Streptococcaceae are relatively small, with between 1.8 and 2.5 Mb, and to date (November 2011), complete genome sequences of the two L. lactis ssp. lactis and cremoris, as well as 13 Streptococcus species, are available. Examination of lactococcal and streptococcal genomes has provided insight into their phylogenetic relationships and has, importantly, enabled analysis of the evolutionary paths followed by these bacteria. Analysis of phylogenetic patterns of Lactobacillales COGs, together with the phylogenetic tree of this order (Fig. 1), suggests that many of the changes that occurred in Streptococcaceae after their divergence from the Bacillus ancestor are related to the transition to new nutrient-rich environments such as milk and the GI tract (Makarova et al., 2006; Makarova & Koonin, 2007).

Genome reduction

Striking in the lactococcal and streptococcal genome sequences is the indication of reduced biosynthetic capacities of these bacteria, likely resulting from genome reduction events as they adapted to new nutrient-rich environments. During this process, a number of genes for the biosynthesis of cofactors, such as heme, have been lost, although this is in some cases compensated for by uptake systems. Dairy L. lactis strains and particularly those with the ‘cremoris’ phenotype have smaller chromosomes than wild-type strains (Kelly et al., 2010). Dairy strains have been selected as essential components of industrial processes and have evolved accordingly, largely through the loss of function, so that they are no longer fit to survive outside the dairy environment. Particular to adaptation to dairy environments, many of the amino acid biosynthetic genes in L. lactis strains are inactivated or do not produce the amino acids in sufficient amounts to support growth (van Niel & Hahn-Hägerdal, 1999). Most L. lactis ssp. lactis dairy isolates are auxotrophic for histidine and branched-chain amino acids (BCAA), while most nondairy isolates are prototrophic for histidine and BCAAs. The inactivation of genes for the biosynthesis of these amino acids has therefore been proposed to have occurred during the adaptation to milk (Delorme et al., 1993; Godon et al., 1993). Although the biosynthetic operons for histidine and BCAA are present, mutations such as the nonsense mutations and deletions identified in the leu genes in L. lactis IL1403 disrupt leucine biosynthesis in this strain (Godon et al., 1993).

The high degree of amino acid auxotrophy observed for L. lactis dairy strains is in contrast to the extensive amino acid biosynthetic capability of S. thermophilus. This dairy bacterium is only auxotrophic for histidine and methionine or cysteine (Pastink et al., 2009). The operon for histidine biosynthesis is absent, and a truncated yhcE gene may contribute to the need for sulfur-containing amino acids (Pastink et al., 2009). In many organisms, TCA enzymes can also have roles in the synthesis of certain amino acid precursors (Fig. 6). This has been observed in S. mutans where glutamate and hence glutamine are synthesized from oxaloacetate (Cvitkovitch et al., 1997) (Fig. 6). The S. pneumoniae R6 genome, on the other hand, contains none of the eight genes comprising the TCA cycle. Streptococcus pneumoniae R6 is therefore incapable of synthesizing aspartate, and therefore lysine, methionine, threonine, and isoleucine from oxaloacetate, nor can it synthesize glutamate, and hence arginine, via α-ketoglutarate.

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Figure 6. Proposed oxidative branch of the TCA cycle in Streptococcaceae. PEP and pyruvate can be converted to oxaloacetate via PEP (PEPC) and pyruvate carboxylase (PC), respectively. The citrate pathway can be used to synthesize glutamate and hence glutamine. Oxaloacetate and acetyl-CoA are required for the formation of citrate, a reaction catalyzed by citrate synthase (CS). Citrate is subsequently converted to isocitrate by aconitase (AC) and then to α-ketoglutarate by isocitrate dehydrogenase (ICDH). The final step in glutamate synthesis is catalyzed by glutamate dehydrogenase (GDH) after which glutamine can be synthesized via the glutamine synthetase–glutamate synthase pathway (GOGAT). Through the action of aspartate aminotransferase (ASP-ATase), aspartate is formed from oxaloacetate. Aspartate can then be used in biosynthesis reactions to form other amino acids or nucleotides.

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Adaptation of the family Streptococcaceae and LAB in general to oxygen-poor environments is evident from the absence of catalase and cytochrome oxidase–related genes (Makarova et al., 2006). Also, no LAB species encodes a complete TCA cycle. The extent of genome reduction varies between the species and is often reflected in the ability of the organisms to synthesize certain amino acids. Further variation in genome reduction is evident within L. lactis ssp. where, for example, L. lactis ssp. cremoris strains MG1363 and SK11 do not contain the cit operon for citrate utilization, while L. lactis ssp. lactis IL1403 does. Citrate utilization was used historically to distinguish between L. lactis ssp. but because this phenotype appears to have been unstable and often plasmid-located (Górecki et al., 2011), it was not a reliable marker (Kempler & McKay, 1981).

Genome reduction appears to have been a major factor in the shaping of streptococcal and lactococcal genomes, and all Streptococcaceae species show loss of specific genes as well as pseudogenes (Wegmann et al., 2007; Makarova et al., 2006). Streptococcus thermophilus in particular has many more pseudogenes than the other Streptococcus species sequenced to date. Pseudogenes account for 10% of the 1.8-MB S. thermophilus genome, while for other Streptococcus species this value is between 0.7% and 4.2% (Bolotin et al., 2004). Notably, the genes encoding four of the seven PTS transporters in S. thermophilus are pseudogenes. The adaptation of this bacterium to the dairy environment is further evident from the loss of functionality of many other carbohydrate utilization genes, in line with the limited range of carbohydrates present in milk (Bolotin et al., 2004). Although S. thermophilus is phylogenetically close to pathogenic streptococci, most streptococcal virulence–related genes that are not involved in basic cellular functions are absent from or inactivated in the former.

Reductive evolution during the adaptation to dairy environments is evident in the carbohydrate utilization genes contained by the L. lactis ssp. sequenced so far. The genomes of L. lactis ssp. lactis IL1403 (Bolotin et al., 2001) and KF147 (Siezen et al., 2010), as well as those of L. lactis ssp. cremoris MG1363 (Wegmann et al., 2007) and SK11 (Makarova et al., 2006), have been fully sequenced, representing both plant (KF147) and dairy (the other 3) isolates. The original habitat of L. lactis is generally believed to be plants, and this is evident in the ability of plant isolates to degrade plant sugars that the dairy strains cannot. For example, L. lactis KF147 can utilize fructans, which are the dominant carbohydrate reserve in many plants, and also contains a gene cluster for the degradation of mannose, polymers of which are present in plant cell walls (Siezen et al., 2008). Some L. lactis dairy strains still contain genes or remnants of genes for the utilization of certain plant sugars. For example, carbohydrate utilization experiments with genome sequence information from L. lactis MG1363 have shown that this strain can utilize mannitol and sorbitol (Wegmann et al., 2007; Kelly et al., 2010). Lactococcus lactis MG1363 as well as L. lactis strains IL1403 and SK11 contains cell surface proteins (CscABCD) present in other Gram-positive plant-associated bacteria, supporting the notion of a plant-associated ancestor (Wegmann et al., 2007).

Gene duplication

The adaption of the family Streptococcaceae to specific environmental niches is also evident from genetic gains, for example, through the duplication of genes involved in carbohydrate transport and metabolism, including those encoding PTSs, β-galactosidases and l-lactate dehydrogenases and those involved in peptide or amino acid transport and utilization (Makarova & Koonin, 2007). Growth in nutrient-rich environments appears to have been the major driving force behind the fixation of gene duplications during evolution.

Pathogenic streptococcal species contain a particularly large number of sugar uptake systems, specific to the carbohydrates they may encounter. Streptococcus mutans is potentially capable of metabolizing a wider variety of carbohydrates than any other Gram-positive organism sequenced to date (Ajdic et al., 2002). Genes with (putative) functions in transport and metabolism of cellobiose, fructose, galactose, glucose, β-glucosides, lactose, maltose/maltodextrin, isomaltosaccharides, mannose, melibiose starch, raffinose, ribulose, sucrose, trehalose, and possibly sorbose are found in the genome of S. mutans. The genome of S. mutans UA159 may encode as many as 14 PTSs with a variety of enzyme II domains with specificity for different sugars (Ajdic et al., 2002). Streptococcus mutans is also able to convert several sugar-alcohols, such as mannitol and sorbitol, to glycolytic intermediates (Ajdic & Pham, 2007). Analysis of the genome sequence of S. pneumoniae suggests that this bacterium also has a sugar utilization range wider than other residents of the human upper respiratory tract sequenced to date (H. influenzae and N. meningitidis) (Tettelin et al., 2000; Taniai et al., 2008). Streptococcus pneumoniae is predicted to contain 21 PTS sugar-specific enzyme II complexes with a variety of gene and domain arrangements, which is, relative to genome size, more than twice that in any other sequenced organism (excluding other Streptococcus species) and emphasizes the importance of sugars to the lifestyle of S. pneumoniae. In addition to the PTSs, S. pneumoniae has seven ABC sugar uptake systems, most of which do not have cytoplasmic ATP-binding components encoded with the substrate-binding and membrane components. Like S. mutans, S. pneumoniae contains genes coding for the catabolism of pentitols via the pentose phosphate pathway (Tettelin et al., 2001; Ajdic et al., 2002). The ability to use a wide variety of carbohydrates is important for the virulence of pathogenic streptococci, allowing these bacteria to persist and effectively compete in their particular environmental niches (Tettelin et al., 2001; Ajdic et al., 2002; Abranches et al., 2008).

Many Lactobacillales species carry two enolase genes, while most bacteria have a single gene for this glycolytic enzyme. Duplicate enolase genes are present in the genomes of L. lactis ssp. lactis strains IL1403 and KF147 and in L. lactis ssp. cremoris SK11, while in L. lactis ssp. cremoris MG1363, enoB is a pseudogene (Wegmann et al., 2007). Both enolases in L. lactis IL1403 have enzymatic activity (Jamet et al., 2001) but their specific physiological functions remain unclear. In pathogenic bacteria such as Brucella abortus (Han et al., 2011) and Streptococcus suis (Esgleas et al., 2008), enolase has been shown to be surface located and involved in human–pathogen interactions. The duplicate enolases in some LAB species may therefore have functions separate to glycolysis. Phylogenetic analysis of the duplicate enolase genes of lactobacilli suggests that one of the gene copies is the ancestral version in Gram-positive bacteria, while the other copy was acquired by the Lactobacillales ancestor from a different bacterial lineage, likely Actinobacteria (Makarova et al., 2006). Lactococcus lactis also contains three chromosomally encoded LDH genes – ldh, ldhB, and ldhX. LdhB activity was observed in an ldh-deletion strain. The ldhB gene, normally silent, could be activated by the insertion of an IS905-like element, creating a hybrid active promoter upstream of ldhB (Gaspar et al., 2007). Duplications of the enolase and/or lactate dehydrogenase genes are not present in the sequenced genomes of Streptococcus species.

The amino acid biosynthetic deficiency in Streptococcaceae is offset by scavenging for resources from the environment using many amino acid and peptide uptake systems. The genome of S. pyogenes encodes for six ABC transporters putatively identified as amino acid uptake systems, as well as two additional transporter systems that appear to mediate the uptake of dipeptides and oligopeptides (Ferretti et al., 2001). Adaptation to using extracellular proteins or peptides is well documented for L. lactis and is discussed below.

Plasmid-borne genes and horizontal gene transfer

As already mentioned, many dairy L. lactis strains carry multiple plasmids representing an important fraction of the genome. In addition to the metabolic functions already named, lactococcal plasmids specify protease activity, exopolysaccharide production, bacteriocin production and immunity, and bacteriophage resistance which is especially important for industrial-scale fermentations (Siezen et al., 2005).

Four plasmids from the dairy isolate L. lactis ssp. cremoris SK11 carry at least nine regions that could have been acquired through horizontal gene transfer (HGT) from enterococci, streptococci, or lactobacilli (Siezen et al., 2005). The plasmid sequences reflect adaptations related to growth on milk components such as lactose and caseins, as well as reveal the presence of genes involved in the uptake of cations such as Mg2+, Mn2+, and possibly Fe2+.

HGT events have also allowed L. lactis to inhabit its original plant environment. Genome sequencing of the plant isolate L. lactis KF147 revealed the presence of several putative transposons (Siezen et al., 2010). The largest putative transposon contains a gene cluster (sucP-agaABCR-galTK-aga-galR-fbp) for α-galactoside utilization, the products of which have very high similarity (92% amino acid sequence identity) to those of L. raffinolactis ATCC43920 (Boucher et al., 2003). The α-galactosidase enzyme, encoded by aga, catalyzes the release of d-galactose moieties from typical plant oligosaccharides such as stachyose, raffinose, and melibiose, making them available for utilization via the Leloir pathway. In both L. lactis KF147 and L. raffinolactis, this gene cluster has a higher than average G+C content, suggesting that it has been acquired by HGT (Siezen et al., 2008). Conjugative transfer of this transposon from L. lactis KF147 to the dairy isolate L. lactis NZ4501 has been achieved in vitro (Siezen et al., 2011). This genetic element is normally not present in the dairy-derived strains and is spontaneously lost upon prolonged growth of L. lactis KF147 in milk (Bachmann, 2009).

Milk contains very low amounts of free amino acids and peptides, and L. lactis ssp., having low amino acid biosynthetic capabilities, may depend on a proteolytic system that allows the degradation and utilization of the predominant protein in bovine milk – casein (Mills & Thomas, 1981; Juillard et al., 1995). Casein contains all the amino acids necessary for the growth of L. lactis. The proteolytic system consists of an extracellular, cell-wall-anchored proteinase (PrtP) that degrades casein into smaller peptides, transport systems that translocate these peptides into the cell, and peptidases that degrade the peptides intracellularly (Tynkkynen et al., 1993). PrtP degrades casein into more than a hundred different oligopeptides ranging from 4 to 30 amino acid residues, which are then transported into the cell by the OppABCDF ABC transporter (Kunji et al., 1998). Lactococcus lactis-derived strains contain a number of different peptidases (at least 9) for the degradation of casein peptides, and many have overlapping activities (Christensen et al., 1999; Liu et al., 2010). Many of the genes or gene clusters for these proteins/enzymes are plasmid-borne (Siezen et al., 2005). In L. lactis SK11, oppABCDF and pepO are located on one plasmid, while prtP is on another (Siezen et al., 2005). During the plasmid curing procedures used to produce the strains L. lactis IL1403 (Chopin et al., 1984) and L. lactis MG1363 (Gasson, 1983), some of these genes including the essential proteinase PrtP were lost. As a result, these bacteria cannot be grown in milk anymore. It has been proposed that in L. lactis dairy isolates, proteases and peptidases provide a selective advantage when growing in milk and this proteolytic system is also found on the chromosome of S. thermophilus LMD9 (Liu et al., 2010). The genes are, however, not restricted to dairy isolates, and some plant-derived L. lactis strains have the largest set of proteolytic system genes (Liu et al., 2010). Subtle changes in the substrate-binding site(s) can change the substrate specificity of lactococcal PrtP proteinases (Exterkate et al., 1993), and it is therefore possible that the proteolytic system also allows L. lactis plant isolates to utilize proteins found in their specific environmental niche. Sequencing of more L. lactis plant isolates as well as investigation into the cleavage specificity of the proteinases may indicate whether the PrtP proteinases present in these strains differ in their substrate-binding regions and whether the switch to dairy environments has resulted in the adaptation of PrtP proteinases to casein degradation.

Other HGT events have allowed S. thermophilus to adapt to the dairy environment. Streptococcus thermophilus possesses a 3.6-kb chromosomal region with extensive similarity to genes from Lactobacillus bulgaricus, which enables it to synthesize methionine (Bolotin et al., 2004). There is a high level of sequence similarity (95%) between both species suggesting a recent HGT event. The two species are rather distantly related. They are used together in yoghurt manufacture and have been observed to adhere to each other (Bolotin et al., 2004). Close association could facilitate the gene transfer between S. thermophilus and L. bulgaricus. The S. thermophilus chromosome also contains regions with very high similarity to the genomes of L. lactis ssp. lactis and L. lactis ssp. cremoris (Bolotin et al., 2004). These could also have been transferred between the bacteria owing to their close ecological proximity.

Adaptation to a pathogenic lifestyle is, in general, accompanied by genome reduction, and pathogens often have small genomes. This results in the evolution of strictly host-dependent bacteria as the bacteria rely on the host cell to compensate for lost gene functions. Genome reduction, however, can be coupled to the acquisition of genomic islands (GIs) and in particular pathogenicity islands (PAIs) via HGT. PAIs often contain functional genes related to drug resistance, immunity, virulence, and metabolism (reviewed in Dobrindt et al., 2004). Newly acquired PAIs have been linked to recent outbreaks of S. suis in China (Chen et al., 2007). An 89-kb PAI was identified, encoding a zeta-toxin, a two-component signal transduction system, three ABC transporter cassettes, and components of a type IV secretion system, which has been demonstrated to mediate HGT of the PAI and contribute to virulence (Li et al., 2011; Zhao et al., 2011).

In bovine-specific S. agalactiae, eight GIs were identified. One GI included an operon involved in the production and immunity of the lantibiotic salivaricin A, with high similarity (94.8%) to genes from S. pyogenes (Richards et al., 2011). In addition, another GI-containing operon for fructose and lactose utilization exhibits high similarity (99%) to a contiguous sequence in Streptococcus dysgalactiae ssp. dysgalactiae (Richards et al., 2011). The presence of these GIs indicates that not only has the genome of mastitis-causing S. agalactiae strains been shaped by HGT, but specifically by HGT from other streptococcal species that cause mastitis. The ability to utilize lactose and fructose might facilitate the survival of S. agalactiae in both mammary and nonmammary organs such as the rumen (Richards et al., 2011). Streptococcus pneumoniae has also adapted to use carbon and nitrogen sources specific to the host tissue it invades. In addition to sugars and amino acids or peptides, polysaccharides and hexosamines are important potential nutritional sources for S. pneumoniae. The genome of S. pneumoniae encodes extracellular enzymatic pathways utilizing N-acetylglucosaminidases, α- and β-galactosidases, endoglycosidases, hydrolases, hyaluronidases, and neuraminidases, enabling the degradation of host mucins, glycolipids, and hyaluronic acid and the bacterium's own capsule (Ferretti et al., 2001; Tettelin et al., 2001). The enzymes increase the substrate availability to S. pneumoniae and also contribute to the virulence of this species, damaging host tissue and facilitating colonization. The presence of extracellular proteases in S. mutans contributes to virulence as well as to nutrition (Cowman et al., 1979; Rosengren & Winblad, 1976). Notwithstanding this, all amino acid biosynthetic pathways were identified in the genome of this bacterium, and it may thus be able to synthesize all of the amino acids (Ajdic et al., 2002).

Competence in Streptococcaceae

It has been demonstrated that L. lactis is able to acquire genetic material through conjugation and bacteriophage transduction (Gasson, 1983; Gasson et al., 1995; Machielsen et al., 2011). Competence-mediated DNA uptake is yet another mechanism for HGT occurring between bacteria. Natural competence has been well studied in S. pneumoniae and S. thermophilus (Havarstein et al., 1997; Cvitkovitch, 2001), but other streptococci such as Streptococcus aginosus, S. mitis, and S. mutans have also been shown to be naturally transformable (Havarstein et al., 1997; Martin et al., 2006). Interestingly, the discovery of DNA as the hereditary material of life was because of the experiments carried out with S. pneumoniae. The observation that heat-treated virulent strains did not kill mice but a mixture of heat-treated virulent and live nonvirulent S. pneumoniae strains did by Griffith in 1928 was further investigated by Avery and led to the identification of DNA as the ‘transforming principle’ in the virulence experiments (see O'Connor (2008) for a review of the early experiments).

Competence development in streptococci relies on the transcriptional induction of comX, which encodes the alternative sigma factor σx. In S. pneumoniae, competence development depends on an early step involving a quorum-sensing system encoded by comABCDE (Claverys & Havarstein, 2002). The inducer peptide, encoded by comC, is secreted into the extracellular medium by the ABC-type transporter encoded by comAB (Alloing et al., 1998). The expression of comC increases with cell density, and once its extracellular concentration reaches a critical threshold, the inducer peptide activates the membrane-located histidine kinase ComD, which in turn stimulates the phosphorylation of the response regulator ComE (Cheng et al., 1997). Phosphorylated ComE positively regulates the expression of comABCDE as well as comX (Pestova et al., 1996). The alternative sigma factor σx associates with the RNA polymerase, allowing synthesis of the late competence genes. The late genes encode the DNA uptake machinery, single-stranded DNA-binding proteins as well as proteins essential for homologous recombination (Lee & Morrison, 1999). This cascade also induces competence in S. mutans where the early competence genes are blpABCHR (Li et al., 2002). In S. thermophilus and S. salivarius, however, induction of σx expression relies on another quorum-sensing system involving the transcriptional regulator ComR and a small hydrophobic peptide pheromone encoded by comS (Fontaine et al., 2010). The Ami or Opp oligopeptide transporter plays an essential role in competence development in S. thermophilus by reimporting secreted, matured ComS (Gardan et al., 2009; Fontaine et al., 2010). ComS is then postulated to bind to ComR which in turn binds to the comX promoter, promoting σx expression (Fontaine et al., 2010).

Another major difference between competence development in S. pneumoniae and S. thermophilus is that while S. thermophilus strain LMD-9 can spontaneously turn on competence in chemically defined medium, competence induction in S. pneumoniae appears to be stress related (Gardan et al., 2009; Claverys et al., 2006). Even though many streptococci are naturally competent, competence is not a constant property. Rather, it is a transient state regulated by the quorum-sensing mechanisms described above. Yet to be identified optimal conditions for competence may partly explain why, although the genome sequences of many streptococci contain competence genes, not all have been shown to be naturally competent.

Genes for competence development have been identified in L. lactis strains, but none of these strains have been naturally transformed. While ComX has been identified and shown to regulate the expression of late competence genes, there is a high occurrence (50% of strains tested) of a mutated dprA gene in L. lactis ssp. lactis strains, including L. lactis ssp. lactis IL1403 (Wydau et al., 2006). dprA (smf in B. subtilis and L. lactis ssp. cremoris) encodes for a DNA-processing protein, which has been shown to play an essential role in the fate of DNA taken up during competence in S. pneumoniae (Bergé et al., 2003). In an S. pneumoniae dprA mutant, the incoming DNA was degraded and DNA fragments were randomly integrated into the chromosome (Bergé et al., 2003). Four different mutations have been identified in the dprA genes of L. lactis ssp. lactis strains, all of which lead to a premature stop codon, inactivating the protein (Wydau et al., 2006). In this background, L. lactis may use the DNA uptake machinery to provide DNA to be degraded, increasing the intracellular pool of nucleotides. This has been observed in E. coli (Finkel & Kolter, 2001), and as free bases are present in only limited amounts in milk, DNA uptake may provide a growth advantage to L. lactis.

Future perspectives

  1. Top of page
  2. Abstract
  3. Introduction
  4. Central metabolism
  5. The effect of oxygen on metabolism of Streptococcaceae
  6. Proton motive force generation by the F1Fo ATPase and solute transport
  7. ADI pathway
  8. Evidence from genome sequences for adaptation of Streptococcaceae to nutrient-rich environments
  9. Future perspectives
  10. References

The rapid and increasingly cost-effective development of whole-genome sequencing now allows the in-depth analysis of many L. lactis ssp. and Streptococcus species and serotypes rather than type strains only. Sequencing of Streptococcaceae important to the dairy industry has revealed that the switch to nutritionally rich environments has been instrumental in the shaping of these small genomes and, importantly, the absence of virulence genes in the genome S. thermophilus has cemented its GRAS status.

The molecular mechanisms underlying the switch from homolactic to mixed-acid fermentation in L. lactis are still unresolved. Because the end products of fermentation have an important influence on the flavor of fermented food products, manipulation of flux through these pathways would allow a targeted approach to flavor formation in products such as cheese and yoghurt. Resequencing of industrial strains would also contribute to the elucidation of specific pathways that contribute to the desirable and nondesirable flavors in existing dairy products.

Exploitation of the natural diversity in carbohydrate and protein utilization systems in lactococci may allow for the development of new, possibly nondairy, fermented food products. Also, engineering of existing acid tolerance systems and cold-shock proteins in dairy Streptococcaceae may produce more robust starter cultures.

Beyond this, the wealth of information generated with regard to virulence genes of pathogenic and nonpathogenic Streptococcus serotypes sequenced to date will lead to more targeted treatments of the numerous life-threatening diseases caused by these bacteria.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Central metabolism
  5. The effect of oxygen on metabolism of Streptococcaceae
  6. Proton motive force generation by the F1Fo ATPase and solute transport
  7. ADI pathway
  8. Evidence from genome sequences for adaptation of Streptococcaceae to nutrient-rich environments
  9. Future perspectives
  10. References